Radiosensitizing effect of echitamidine-N-oxide-19-ο-β-D-glucopyranoside isolated from Alstonia scholaris in KB cells

The ability of echitamidine-N-oxide-19-ο-β-D-glucopyranoside (EOG) that has been isolated from the stem bark of Alstonia scholaris to enhance the effect of radiation was studied in KB cells exposed to different doses of γ-rays. Treatment of KB cells with 1, 5, 10, 20, 30, 40, 50, or 100 µg/ml EOG for 1–6 h before 2 Gy of γ-radiation caused a concentration and time-dependent depletion in the clonogenicity of cells, giving a EOG-induced clonogenic cytotoxicity half maximal inhibitory concentration (IC50) of 20 µg/ml for a 6 h treatment, whereas the lowest survival of 0.29 was observed for 100 µg/ml EOG treated for 6 h before 2 Gy exposure. In a separate experiment, exposure of KB cells to 0, 0.25, 0.5, 1, 2, 3, or 4 Gy of γ-radiation led to a dose-dependent reduction in the clonogenicity of cells, whereas treatment of KB cells with 20 µg/ml of EOG one hour before irradiation to 0 to 4 Gy further attenuated their clonogenicity. The micronuclei assay revealed a dose-dependent rise in their frequency at 20, 30, and 40 h post-irradiation, and 20 µg/ml EOG increased the radiation-induced micronuclei (p < 0.005), which reached maximum at 30 h post-irradiation in both groups. This rise in micronuclei was approximately threefold at 0.5 Gy, 2.5-fold for 1 and 2 Gy, and 1.5-fold for 3 and 4 Gy irradiation. The biochemical estimations showed that irradiation of KB cells to 2 Gy increased the lactate dehydrogenase activity, which was significantly enhanced by EOG pretreatment. Similarly, a rise in lipid peroxidation was discernible after irradiation, and EOG treatment significantly raised it compared to irradiation alone. In contrast, irradiation of KB cells to 2 Gy γ-radiation reduced the glutathione concentration, whereas EOG pretreatment attenuated it significantly. The present investigation demonstrates that EOG augments the cytotoxic effect of radiation in KB cells by increasing the damage to the cellular genome, lipid peroxidation, and lactate dehydrogenase and depleting the glutathione level.